Chitosan hydrogel derivatives as a coating agent with broad spectrum of antimicrobial activities

ABSTRACT

The present invention relates to water soluble quaternized chitosan derivatives which form hydrogel matrix with broad antimicrobial properties for the protection and coating of medical device. Hydrogel is attractive as an antimicrobial coating since its hydrophilicity intrinsically prevents the reversible nonspecific attachment of microbes. In order to achieve hydrogel formation, quaternized chitosan can be grafted with polymerizable groups, especially photocrosslinkable groups, such as methacrylates, PEG derivatives and be converted into hydrogels through a thermal or UV polymerization process. Hydrogels are hydrated cross-linked polymeric systems that contain water in an equilibrium state forming cushion water shield. The present invention is widely used in many medical devices. This invention describes the formation of novel hydrogels based on quaternized ammonium chitosan derivatives which has been grafted with photocrosslinkable groups, hence providing hydrogels as antimicrobial water shield coating agent.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/383,684 entitled “Chitosan Hydrogel Derivatives as a CoatingAgent with Broad Spectrum of Antimicrobial Activities,” filed Mar. 15,2012. U.S. patent application Ser. No. 13/383,684 is a 35 USC §371 ofPCT/JP2009/063013, filed Jul. 13, 2009. Each of these applications ishereby incorporated by reference in their entirety.

BACKGROUND ART

Chitosan is a β-1,4-linked polymer of glucosamine(2-amino-2-deoxy-β-D-glucose) and lesser amounts of N-acetylglucosamine.It is formed by the deacetylation of chitin (poly-N-acetylglucosamine),an abundant byproduct of the crab and shrimp processing industries.

The water solubility and antimicrobial property of native chitosan canbe achieved or enhanced via the quaternization of chitosan. Quaternizedchitosan, as a consequence of the quaternization of the amino group inthe C-2 position, gained a permanent positive charge on thepolysaccharide backbone. Quaternized chitosans find utility in cosmeticpreparations, food preservation and packaging, coating materials,disinfectants and biomedical applications (JP-A-2005-290297,JP-A-8-144121, JP-A-2004-515813).

Hence, quaternized chitosan can be utilized for hydrogel formation andformulated as antimicrobial contact lens coating. In order to achievehydrogel formation, quaternized chitosan can be grafted withphotocrosslinkable groups, such as (meth)acrylates, and be convertedinto hydrogels through a UV polymerization process. Hydrogels arehydrated cross-linked polymeric systems that contain water in anequilibrium state and have been widely used in many biomedicalapplications including contact lenses, bio-implants such as urinarycatheters, pacemaker, heart valves, artificial heart, mammaryprosthesis, intraocular lenses, wound dressings, artificial organs anddelivery carriers for the bioactive reagents due to their high degree ofbiocompatibility. However, there have been no studies on the use ofquaternized chitosan hydrogels as antimicrobial medical device coating.The present invention describes the formation of novel hydrogels basedon quaternized chitosan which has been grafted with polymerizable group,especially photocrosslinkable group, and relates to medical devicehaving resultant antimicrobial activities.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a novel and usefulquaternized chitosan derivative, a method of producing the quaternizedchitosan derivative, a chitosan hydrogel, a method of producing thehydrogel, and an article using the hydrogel.

The present invention provides following embodiments:

-   [1] A quaternized chitosan derivative having polymerizable organic    moieties.-   [2] The quaternized chitosan derivative of [1], having an ammonium    group represented by the following formula (1):    —N⁺(R¹)₃   (1)    where each R¹ is independently selected from the group consisting of    hydrogen and a substituted or unsubstituted alkyl group, two or    three of (R¹)s may combine to form a substituted or unsubstituted    aliphatic cyclic group.-   [3] The quaternized chitosan derivative of [2], wherein at least one    of (R¹)s is a substituted or unsubstituted alkyl group having 1 to    18 carbon atoms.-   [4] The quaternized chitosan derivative of any one of [1] to [3],    wherein the polymerizable organic moiety is represented by the    following formula:    —Y—P

(where P is a polymerizable group; Y is a spacer group).

-   [5] The quaternized chitosan derivative of [2], wherein said    —N⁺(R¹)₃ group is —N⁺R′R″₂ where R′ and R″ are independently    selected from the group consisting of hydrogen and a substituted or    unsubstituted alkyl group, provided that R′ has more carbon atoms    than R″.-   [6] A method of producing a quaternized chitosan derivative having    polymerizable organic moieties, comprising a step of reacting a    quaternized chitosan derivative and a halide having a polymerizable    organic moiety.-   [7] The method of [6], wherein said halide is represented by the    following formula (2):    X—P¹   (2)    where P¹ is a polymerizable organic moiety; X is a halogen atom.    Preferred examples of X include Cl, Br and I. Preferred examples of    the halide X—P¹ include X-Z-COOCR⁴═CH₂ where R⁴ is —H or —CH₃; Z is    a chemical bond or a polyethylene glycol chain-containing group such    as —CH₂COO—(CH₂CH₂O)_(n)— (n is 1 or more and preferably is 3 or    more.).-   [8] A method of producing a chitosan hydrogel, comprising a step of    polymerizing a polymerizable composition comprising said quaternized    chitosan derivative of any one of [1] to [5], initiator, and water.-   [9] A method of producing a chitosan hydrogel of [8], wherein said    polymerizing step comprises a step of irradiating ultraviolet (UV)    to a photopolymerizable composition comprising said quaternized    chitosan derivative of any one of [1] to [5], photoinitiator, and    water. Preferred polymerizable (especially, photopolymerizable)    composition comprises 0.1 to 20% by weight of said quaternized    chitosan derivative of any one of [1] to [5], 0.1 to 1.0% by weight    of initiator (especially, photoinitiator), 1 to 99.9% by weight of    one or more comonomers, and 0 to 80% by weight of water per the    total amount of the composition. The composition may contain other    components, provided that the advantage of the present invention is    not impaired.-   [10] A hydrogel produced by the method of [8] or [9].-   [11] A medical device having a coating comprising the hydrogel of    [10].-   [12] A contact lens having a coating comprising the hydrogel of    [10].-   [13] A medical device containing the hydrogel of [10].-   [14] A cosmetic containing the hydrogel of [10].

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows scanning electron micrographs of several chitosan basedhydrogels (Example 15). FIG. 1( a) corresponds to unquaternized chitosanhydrogel; FIG. 1( b) corresponds to trimethylammonium chitosan-g-PEGMAhydrogel; FIG. 1( c) corresponds to trihexylammonium chitosan-g-PEGMAhydrogel; FIG. 1( d) corresponds to tridecylammonium chitosan-g-PEGMAhydrogel; FIG. 1( e) corresponds to dimethylhexylammoniumchitosan-g-PEGMA hydrogel; and FIG. 1( f) corresponds todimethyldecylammonium chitosan-g-PEGMA.

FIG. 2 shows results of Atomic Force Microscope (AFM) observationreveals that trimethylammonium chitosan-g-PEGMA hydrogel was coated onthe surface of silicon-containing rigid gas permeable contact lenses(RGP CLs).

DESCRIPTION OF EMBODIMENTS

The present invention relates to water soluble quaternized chitosanderivatives which form hydrogel matrix with broad antimicrobialproperties for the protection and coating of medical device. Hydrogel isattractive as an antimicrobial coating since its hydrophilicityintrinsically prevents the reversible nonspecific attachment ofmicrobes.

In order to achieve hydrogel formation, quaternized chitosan can begrafted with polymerizable groups, especially photocrosslinkable groups,such as methacrylates, PEG derivatives and be converted into hydrogelsthrough a thermal or UV polymerization process. Hydrogels are hydratedcross-linked polymeric systems that contain water in an equilibriumstate forming cushion water shield. The present invention is widely usedin many medical devices for example biomedical devices such as contactlenses, bio-implants such as urinary catheters, pacemaker, heart valves,artificial heart, mammary prosthesis, intraocular lenses, wounddressings, artificial organs and delivery carriers for the bioactivereagents, total joint replacement due to their high degree ofbiocompatibility, antimicrobial property and good wettingcharacteristics. This invention describes the formation of novelhydrogels based on quaternized ammonium chitosan derivatives which hasbeen grafted with polymerizable groups, especially photocrosslinkablegroups, hence providing hydrogels as antimicrobial water shield coatingagent.

The present invention will now be further described. In the followingpassages, different aspects of the invention are defined in more detail.Each aspect so defined may be combined with any other aspect or aspectsunless clearly indicated to the contrary. In particular, any featureindicated as being preferred or advantageous may be combined with anyother feature or features indicated as being preferred or advantageous.

In a first, highly preferred embodiment, the inventive antimicrobialhydrogels comprise a crosslinked hydrogel prepared by the free radicalpolymerization of a polymerizable cationic quaternized chitosan.

A schematical structure of the polymerizable cationic quaternizedchitosan is shown in following formula (3):

where P is a polymerizable group; Y is a spacer group; and R′ and R″ areindependently selected from the group consisting of hydrogen and asubstituted or unsubstituted alkyl group. Formula (3) is illustrated foronly explanation of the present invention. It should be noted that thepolymerizable quaternized chitosan of the present invention is notlimited to an embodiment of formula (3).

The cationic quaternized chitosan is preferably prepared from aquaternised chitosan derivative, although other cationic quaternizedchitosans may be used. These cationic quaternized chitosans containtrialkylammonium groups along the quaternized chitosan chain. Thequaternized chitosan will also contain a polymerizable group attached tothe quaternized chitosan chain via a primary hydroxyl group, preferablyvia a spacer group Y.

Referring to formula (3), preferred embodiments of the cationicquaternized chitosan will comprise a quaternized chitosan containingtrialkylammonium groups along the polymer chain, wherein all three alkylgroups are identical. Non-limiting Examples of the quaternized ammoniummoiety include trimethylammonium-, triethylammonium-,tripropylammonium-, tributylammonium-, tripentylammonium-,trihexylammonium-, trioctylammonium-, tridecylammonium-,tridodecylammonium-, trioctadecylammomium-.

More preferred are trialkylammonium moieties wherein one alkyl groupdiffers from the other two. Non-limiting Examples of this more preferredembodiment include dimethylethylammonium-, dimethylpropylammonium-,dimethylbutylammonium-, dimethylpentylammonium-, dimethylhexylammonium-,dimethyloctylammonium-, dimethyldecylammonium-,dimethyldodecylammonium-,dimethyloctadecylammomium-dihexadecylmethylammonium-,dihexadecylethylammonium-, dihexadecylpropylammonium-, anddihexadecylbutylammonium-.

Again, referring to formula (3), the polymerizable group P may compriseone of the polymerizable groups selected from the group comprisingvinyl, acrylate, methacrylate, vinyl phenylene, cinnamoyl, allyl. Thepolymerizable group P is preferably attached to the cationic quaternizedchitosan via a spacer group Y. The spacer group may be selected from thegroup comprising —(C_(n)H_(2n))—, or more preferably —(C_(n)H_(2n)O)—,wherein n=0 to 20, or more preferably between 2 and 10.

In a preferred embodiment, the hydrogel is produced by the free radicalpolymerization of the polymerizable cationic quaternized chitosan. Inanother preferred embodiment, the polymerizable cationic quaternizedchitosan may be copolymerized with a comonomer selected from thenon-limiting group comprising acrylic acid, methyl acrylate, ethylacrylate, hydroxyethyl acrylate, hydroxypropyl acrylate, glycerolmonoacrylate, acryloylphosphorylcholine, polyethylene glycol diacrylate,methacrylic acid, methyl methacrylate, ethyl methacrylate, hydroxyethylmethacrylate, hydroxypropyl methacrylate, glycerol monomethacrylate,methacryloylphosphorylcholine, polyethylene glycol di(meth)acrylate,styrene and N-vinyl pyrrolidinone, or mixtures thereof. The examples ofthe comonomer also include difunctional or trifunctional monomers suchas ethylene glycol diacrylate, glycerol diacrylate, glyceroltriacrylate, divinylbenzene, polyethylene glycol diacrylate, ethyleneglycol di(meth)acrylate, glycerol di(meth)acrylate, glyceroltri(meth)acrylate, divinylbenzene, polyethylene glycol di(meth)acrylate,glycidyl acrylate, glycidyl(meth)acrylate, polypropylene glycoldi(meth)acrylate, polyoxyethylene-polyoxypropylene blockcopolymer andmixtures thereof.

Heretofore, trialkylammonium chitosan can be synthesized throughreacting with alkyl halide such as methyl iodide, hexyl bromide or decylbromide in a nucleophilic substitution reaction with the resultantformation of the quaternized structure. See following reaction scheme I.In the present invention, preferred starting materials include chitosanpowder with a degree of deacetylation of preferably at least 70%, morepreferably at least 75%, and preferably number average molecular weightof 10,000 to 200,000 g/mol (determined through GPC). When the molecularweight of the starting chitosan material is excessively high, it isdifficult to polymerize the chitosan derivative of the presentinvention. In this event, fixation of a hydrogel to a surface of asubstrate would also be difficult.

N-alkyl-N,N-dialkylammonium chitosan can also be formed by firstreductively alkylating chitosan with aldehydes (such as hexanal anddecanal) to form imines, followed by reduction to obtain N-alkylderivatives. The alkyl derivative was then quaternized with alkyl halidesuch as methyl iodide. See following reaction scheme II.

In order to be applied as hydrogel, the quaternized chitosan derivativeswas subsequently grafted with polymerizable groups, especiallyphotocrosslinkable groups and converted to hydrogels via a thermal or UVpolymerization process. The photocrosslinkable group used here ispolyethylene glycol monoacrylate (PEGMA). Chitosan and the quaternizedchitosan were each grafted with PEGMA (of a low molecular weight of375), leading to the formation of chitosan-g-PEGMA and quaternizedchitosan-g-PEGMA. Chitosan-g-PEGMA and quaternized chitosan-g-PEGMA wererespectively synthesized through reacting chloro-functionalised PEGMAwith native chitosan or quaternized chitosan, using sodium hydroxide asbase. After grafting reaction, the water solubility of the quaternizedchitosans were not changed while chitosan, after grafting with thehydrophilic PEGMA, changed from insoluble to soluble. The presence ofgrafted PEG can be detected by using UV spectroscopy employing a PEGassay.

Polyethylene glycol diacrylate (PEGDA), polyethylene glycoldimethacrylate, polypropylene glycol di(meth)acrylate, orpolyoxyethylene-polyoxypropylene blockcopolymer can be blended with thephotocrosslinkable, quaternized chitosan derivatives during UVirradiation. PEGDA has been widely used in photochemical modificationfor the use of hydrogel scaffold, membrane material and drug delivery.Blending with the photocrosslinkable PEGDA has several advantages due toits hydrophilicity, biocompatibility and biodegradability. In addition,PEG being an uncharged polymer, has minimal interaction with itself andother molecules. The most important reason is that by blending withPEGDA, the tensile strength of the chitosan derivatives, in the currentinvention, can be improved, resulting in hydrogels which can be easilyhandled.

In this invention, photopolymerisation was used to produce hydrogels dueto its many advantages. Photopolymerisation is a versatile and faciletechnique to produce water-insoluble hydrogels from liquids. It is alsoa non-invasive technique where hydrogels can be formed in a short timeunder ambient temperature without any detrimental effects to thehydrogels' components. Low organic solvent level was also involved inthe process and the use of toxic chemical crosslinkers (in conventionalchemical crosslinking) such as epichlorohydrin, glutaraldehyde,carbodiimide and so on are avoided. UV light was used to interact withlight sensitive compounds called photoinitiators to create free radicalsthat can initiate polymerization to formed crosslinked hydrogels. Hence,hydrogels can be made from solutions consisting of chitosan-g-PEGacrylate or quaternized chitosan-g-PEG acrylate and PEG diacrylate in2:1 ratio (20 wt % total polymer mixture and 80 wt % water) with smallamount of water-soluble photoinitiator (Irgacure 2959) (0.1 wt % withrespect to the polymer mixture). This will form the fullinterpenetrating network system where the chitosan derivatives werecopolymerized with the PEGDA. After dissolution, around 0.25 ml of eachsolution was added to polypropylene tube and placed in a glass chamberwith argon purge. The sample solution in glass chamber was exposed to UVfor 15 min for hydrogel formation. Alternatively, photopolymerisationcan also be carried out on glass slides with a spacer frame of 250 μmthick. The precursor solution was dropped onto the glass slide, coveredwith another glass slide and then exposed to UV for 15 min, thusproducing the hydrogel.

Larger pore size of freeze-dried polymers can be achieved viaN-alkylation of chitosan. The SEM cross-sectional images for thefreeze-dried polymers of the chitosan and some quaternized chitosan areshown in FIG. 1. FIG. 1 shows that the unquaternized chitosan hydrogelhas many small pores (of a few micrometers) and the quaternized chitosanhydrogels generally have larger pores greater than 100 μm under the samemagnification (×220). Dimethyldecylammonium chitosan hydrogel (seeSynthetic Example 14) was observed to have relatively larger porescompared to dimethylhexylammonium chitosan hydrogel (see SyntheticExample 13). Other applications:

The antimicrobial hydrogels described herein exhibit potentantimicrobial activity against bacteria and fungi, thus they can be usednot only as contact lens coating, but also be included in formulationswhere it is desirable to minimize bacterial attack. For example, theymay be incorporated at low concentrations into cosmetic formulations,such as hair and skin formulations or into contact lens solutions.

These chitosan derivative hydrogels may also applied as biomedical orpharmaceutical materials which can be applied onto or contacted with thebody surface of humans or other animals in order to prevent or inhibitthe proliferation of microorganisms therein.

The antimicrobial hydrogels, prepared from biocompatible materials suchas PEG and chitosan, can also be used in the field of tissueengineering, engaging in 2-dimensional or 3-dimensional cell culture.The antimicrobial hydrogels, due to the presence of positive charge, canalso be used in the delivery and controlled release of negativelycharged bioactive materials such as growth factors or cell adhesiveproteins.

SYNTHETIC EXAMPLES

Chitosan powder was supplied by Dalian Xindie Chitin Co., China with adegree of deacetylation of 81.79% and number average molecular weight of1.05×10⁵ g/mol (determined through GPC). N-methyl-2-pyrrolidinone,sodium iodide, methyl iodide, hexyl bromide, decyl bromide, hexanal,decanal, chloroacetyl chloride, PEG monoacrylate (M_(n) 375), PEGdiacrylate (M_(n) 700), potassium carbonate, sodium hydroxide,isopropanol and sodium borohydride were purchased from Sigma-Aldrich(Saint Louis, Mo., USA). Analytical grade hexane, toluene, diethylether, ethanol, acetone and methylene chloride were purchased from VWRPte Ltd (Singapore) and used as received. Toluene was dried overmolecular sieves. The photoinitiator,4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone (Irgacure 2959) waspurchased from Ciba (Singapore).

¹H-NMR and ¹³C-NMR spectra were recorded with a Bruker Avance 300 MHzinstrument. Fourier Transform Infra Red (FTIR) spectra were recorded atroom temperature using a Digilab FTS 3100 instrument over the wavenumberrange of 4000-400 cm⁻¹ using KBr disks. For Scanning Electron Microscopy(SEM), the hydrogels were immersed in deionised water for 2 days orlonger prior observation. These hydrogels were then frozen at −50° C. inthe freeze dryer for three hours before they were vacuum dried. After 48h of freeze-drying, the hydrogels were cut with a sharp scalpel toobtain the cross-section. They were then mounted onto an aluminum studand sputter-coated with platinum for 60 s using a Jeol JFC-1600 AutoFine coater. The samples were then imaged by scanning electronmicroscope (Jeol JSM-5600LV).

Synthetic Example 1 Preparation of Chlorofunctionalised PEG Acrylate

PEG monoacrylate (2.36 ml, 7.06 mmol), with a number average molecularweight of 375, was dissolved in 100 ml toluene and treated withchloroacetylchloride (2.25 ml, 28.24 mmol). The mixture was then heatedunder reflux for 24 hours. After this time, the solvent and volatileswere removed by evaporation and the residue was dissolved in methylenechloride (150 ml). The solution was stirred over potassium carbonate andfiltered. The solvent was removed through evaporation and after washingwith hexane, the product was obtained and dried, and used withoutfurther purification.

¹H-NMR (300 MHz, CDCl₃), δ: 3.5-3.7 (—O—CH ₂—CH ₂—O—), 4.1 (Cl—CH₂—COO—), 5.7 (—CH═CH₂), 6.0, 6.2 (—CH═CH ₂)

¹³C-NMR (300 MHz, CDCl₃), δ: 63.6-69.0 (—O—CH₂—CH₂—O—), 128, 131(—CH═CH₂), 166 (—OCO—CH═CH₂), 167 (Cl—CH₂—COO—)

Synthetic Example 2 Preparation of Trimethylammonium Chitosan

Chitosan (1 g) was added to N-methyl-2-pyrrolidinone (50 ml) and treatedwith 1.5 N NaOH solution (15 ml). The mixture was stirred for 30 min at50° C. Sodium iodide (1.08 g) and methyl iodide (11.2 g) were then addedto the solution, which was then stirred for 24 hours at 50° C. Thereaction mixture was then filtered to remove the insoluble material, andthe filtrate was then precipitated into a large excess of acetone andfiltered. The resulting product collected by filtration, and dried undervacuum.

¹H-NMR (300 MHz, D₂O), δ: 1.95 (—NHCOCH ₃) 3.33 (—N⁺CH ₃), 3.4-4.1(chitosan-H-2-6)

Synthetic Example 3 Preparation of Trihexylammonium Chitosan

Trihexylammonium chitosan was prepared in the same manner as SyntheticExample 2 except for changing from methyl iodide (11.2 g) to hexylbromide (13.0 g).

¹H-NMR (300 MHz, D₂O), δ: 0.74 (—N⁺CH₂CH₂(CH₂)₃CH ₃), 1.19 (—N⁺CH₂CH₂(CH₂)₃CH₃), 1.25 (—N⁺CH₂CH ₂(CH₂)₃CH₃), 1.95 (—NHCOCH ₃) 3.07 (—N⁺CH₂CH₂(CH₂)₃CH₃), 3.4-4.1 (chitosan-H-2-6)

Synthetic Example 4 Preparation of Tridecylammonium Chitosan

Tridecylammonium chitosan was prepared in the same manner as SyntheticExample 2 except for changing from methyl iodide (11.2 g) to decylbromide (17.4 g).

¹H-NMR (300 MHz, D₂O), δ: 0.74 (—N⁺CH₂CH₂(CH₂)₇CH ₃), 1.16 (—N⁺CH₂CH₂(CH₂)₇CH₃), 1.58 (—N⁺CH₂CH ₂(CH₂)₇CH₃), 1.94 (—NHCOCH ₃), 3.02 (—N⁺CH₂CH₂(CH₂)₇CH₃), 3.4-4.1 (chitosan-H-2-6)

synthetic example 5 Preparation of Chitosan-g-PEGMA

Chitosan (0.2 g) was added to a solution of 50 wt % NaOH solution (2.5ml), and stirred for 24 h. The alkalized chitosan was then filtered, andthe filtrate transferred into a flask. Chlorofunctionalised PEG acrylate(0.5 g, see Synthetic Example 1) was dissolved in isopropanol (2.5 ml)and added dropwise into the chitosan with stirring. The mixture wasreacted for 24 h at room temperature. After this time, the crude productwas recovered by filtration, and dissolved in water (10 ml). Thesolution was neutralised with 2.5M HCl. After centrifugation of thesolution to remove the precipitate and addition of ethanol (40 ml) tothe supernatant, the product precipitated from the solution. The solidwas filtered and rinsed thrice with ethanol.

¹H-NMR (300 MHz, D₂O), δ: 1.94 (—NHCOCH ₃), 3.5-3.7 (—O—CH ₂—CH ₂—O—),3.4-4.1 (chitosan-H-2-6), 5.5 (—CH═CH₂), 6.02 (—CH═CH ₂)

Synthetic Example 6 Preparation of Trimethylammonium Chitosan-g-PEGMA

Trimethylammonium chitosan (0.2 g, see Synthetic Example 2) in 0.45 mlwater was added with 0.30 ml of 0.38 M sodium hydroxide solution.Chlorofunctionalised PEG acrylate (0.5 g, see Synthetic Example 1)dissolved in 1.5 ml isopropanol was then added with stirring and themixture stirred at room temperature for 3 h. After the reaction, themixture was cooled to room temperature and precipitated in a mixture ofacetone and ethanol. The product was obtained after filtration.

¹H-NMR (300 MHz, D₂O), δ: 1.95 (—NHCOCH ₃), 3.22 (—N⁺CH ₃), 3.5-3.7(—O—CH ₂—CH ₂—O—), 3.4-4.1 (chitosan-H-2-6), 5.5 (—CH═CH₂), 5.9-6.1(—CH═CH ₂)

Synthetic Example 7 Preparation of Trihexylammonium Chitosan-g-PEGMA

Trihexylammonium chitosan-g-PEGMA was prepared in the same manner asSynthetic Example 6 except for changing from trimethylammonium chitosan(0.2 g) to trihexylammonium chitosan (0.2 g, see Synthetic Example 3).

¹H-NMR (300 MHz, D₂O), δ: 0.86 (—N⁺CH₂CH₂(CH₂)₃CH ₃), 1.19 (—N⁺CH₂CH₂(CH₂)₃CH₃), 1.30 (—N⁺CH₂CH ₂(CH₂)₃CH₃), 1.95 (—NHCOCH ₃), 3.07 (—N⁺CH₂CH₂(CH₂)₃CH₃), 3.5-3.7 (—O—CH ₂—CH ₂—O—), 3.4-4.1 (chitosan-H-2-6),5.6 (—CH═CH₂), 5.97-6.2 (—CH═CH ₂)

Synthetic Example 8 Preparation of Tridecylammonium Chitosan-g-PEGMA

Tridecylammonium chitosan-g-PEGMA was prepared in the same manner asSynthetic Example 6 except for changing from trimethylammonium chitosan(0.2 g) to tridecylammonium chitosan (0.2 g, see Synthetic Example 4).

¹H-NMR (300 MHz, D₂O), δ: 0.74 (—N⁺CH₂CH₂(CH₂)₇CH ₃), 1.16 (—N⁺CH₂CH₂(CH₂)₇CH₃), 1.58 (—N⁺CH₂CH ₂(CH₂)₇CH₃), 1.94 (—NHCOCH ₃), 3.02 (—N⁺CH₂CH₂(CH₂)₇CH₃), 3.5-3.7 (—O—CH ₂—CH₂—O—), 3.4-4.1 (chitosan-H-2-6), 5.6(—CH═CH₂), 5.91-6.05 (—CH═CH ₂)

Synthetic Example 9 Preparation of N-Hexyl Chitosan

Chitosan (1.0 g, 6.21 mmol) was dissolved in 1% aq. acetic acid (100ml). Hexanal (0.62 g, 0.74 ml, 1 eqv.) was then added, and the mixturestirred at room temperature. After 1 hour of stifling, the pH of thesolution was adjusted to 4.5. To this solution, 10% aq. solution ofsodium borohydride (9.32 mmol) was added and the solution stirred for anadditional 90 minutes. After this time, the pH of the solution wasadjusted to 10, and the precipitated N-hexyl chitosan collected byfiltration, and washed with water until the filtrate was of neutral pH.These precipitants were filtered and the residue washed with distilledwater to neutrality. The unreacted aldehyde and inorganic products weresoxhlet extracted with ethanol and diethyl ether. The resulting N-hexylchitosan was filtered and dried.

Synthetic Example 10 Preparation of N-Decyl Chitosan

N-decyl chitosan was prepared in the same manner as Synthetic Example 9except for changing from hexanal (0.62 g) to decanal (0.97 g, 6.2 mmol).

Synthetic Example 11 Preparation of Dimethylhexylammonium Chitosan

N-Hexyl chitosan (1 g, see Synthetic Example 9) was added toN-methyl-2-pyrrolidinone (50 ml) and treated with 1.5 N NaOH solution(15 ml). The mixture was stirred for 30 min at 50° C. Sodium iodide(1.08 g) and methyl iodide (11.2 g) were then added to the solution,which was then stirred for 24 hours at 50° C. The reaction mixture wasthen filtered to remove the insoluble material, and the filtrate wasthen precipitated into a large excess of acetone and filtered. Theresulting product was collected by filtration, and dried under vacuum.

¹H-NMR (300 MHz, D₂O), δ: 0.76 (—N⁺CH₂CH₂(CH₂)₃CH ₃, 1.22 (—N⁺CH₂CH₂(CH₂)₃CH₃), 1.58 (—N⁺CH₂CH ₂(CH₂)₃CH₃), 1.96 (—NHCOCH ₃), 3.24 (—N⁺CH ₃),3.4-4.1 (chitosan-H-2-6)

Synthetic Example 12 Preparation of Dimethyldecylammonium Chitosan

Dimethyldecylammonium chitosan was prepared in the same manner asSynthetic Example 11 except for changing from N-hexyl chitosan (1 g) toN-decyl chitosan (1 g, see Synthetic Example 10). ¹H-NMR (300 MHz, D₂O),δ: 0.71 (—N⁺CH₂CH₂(CH₂)₇CH ₃), 1.17 (—N⁺CH₂CH₂(CH ₂)₇CH₃), 1.58(—N⁺CH₂CH ₂(CH₂)₇CH₃), 1.94 (—NHCOCH ₃), 3.23 (—N⁺CH ₃), 3.4-4.1(chitosan-H-2-6)

Synthetic Example 13 Preparation of DimethylhexylammoniumChitosan-g-PEGMA

Dimethylhexylammonium chitosan-g-PEGMA was prepared in the same manneras Synthetic Example 6 except for changing from trimethylammoniumchitosan (0.2 g) to dimethylhexylammonium chitosan (0.2 g, see SyntheticExample 11).

¹H-NMR (300 MHz, D₂O), δ: 0.75 (—N⁺CH₂CH₂(CH₂)₃CH ₃), 1.22 (—N⁺CH₂CH₂(CH₂)₃CH₃), 1.58 (—N⁺CH₂CH ₂(CH₂)₃CH₃), 1.96 (—NHCOCH ₃), 3.22 (—N⁺CH ₃),3.5-3.7 (—O—CH ₂—CH ₂—O—), 3.4-4.1 (chitosan-H-2-6), 5.51 (—CH═CH₂),5.86-5.99 (—CH═CH ₂)

Synthetic Example 14 Preparation of DimethyldecylammoniumChitosan-g-PEGMA

Dimethyldecylammonium chitosan-g-PEGMA was prepared in the same manneras Synthetic Example 6 except for changing from trimethylammoniumchitosan (0.2 g) to dimethyldecylammonium chitosan (0.2 g, see SyntheticExample 12).

¹H-NMR (300 MHz, D₂O), δ: 0.74 (—N⁺CH₂CH₂(CH₂)₇CH ₃), 1.22 (—N⁺CH₂CH₂(CH₂)₇CH₃), 1.58 (—N⁺CH₂CH ₂(CH₂)₇CH₃), 1.94 (—NHCOCH ₃), 3.22 (—N⁺CH ₃),3.5-3.7 (—O—CH ₂—CH ₂—O—), 3.4-4.1 (chitosan-H-2-6), 5.54 (—CH═CH₂),5.85-6.05 (—CH═CH ₂)

Preparation of Hydrogels

Synthetic Example 15 Photopolymerisation of Chitosan-g-PEGMA andQuaternized Chitosan-g-PEGMA

Chitosan-g-PEGMA (0.06 g, see Synthetic Example 5) and PEGDA (0.06 g)were blended and dissolved in water (0.48 ml). The photoinitiator(Irgacure 2959) was added at 0.1 wt % (based on the total amount ofChitosan-g-PEGMA, PEGDA and water). The resultant solution was placedinside a mould produced from two glass plates with a 0.25 mm thickspacer frame in between. The solution was then exposed to UV light (365nm, 15 min exposure, 10 mW/cm²) using a Honle UV Technology machineequipped with a UV mercury lamp. After curing, the mould was opened, thehydrogel removed and washed with deionised water.

Similarly prepared were hydrogels produced from trimethylammoniumchitosan-g-PEGMA, trihexylammonium chitosan-g-PEGMA, tridecylammoniumchitosan-g-PEGMA, dimethylhexylammonium chitosan-g-PEGMA anddimethyldecylammonium chitosan-g-PEGMA.

SEM Images of Hydrogels

The SEM cross-sectional images for the freeze-dried polymers of thechitosan and some quaternized chitosan are shown in FIG. 1. FIG. 1 showsthat the unquaternized chitosan hydrogel has many small pores (of a fewmicrometers) and the quaternized chitosan hydrogels generally havelarger pores greater than 100 μm under the same magnification (×220).Dimethyldecylammonium chitosan hydrogel (Synthetic Example 14) wasobserved to have relatively larger pores compared todimethylhexylammonium chitosan hydrogel (Synthetic Example 13).

Application Examples

Antimicrobial Activity

General Culture Procedure

The hydrogels prepared from photopolymerisation were soaked in phosphatesaline buffer overnight and rinsed several times before antimicrobialassay. The bacterium was cultured and harvested for 24 h on TrypticaseSoy Agar before it was washed twice with sterile purified water bycentrifugation. The bacterial suspension (10 μl) in purified water wastransferred to the centre of the hydrogel coating in the well of atissue culture polystyrene plate. The hydrogel was in contact with themicroorganism at 24° C. for 4 hours (exposure time). A small volume ofneutralizing broth was then added to the well to recover any bacterialsurvivors. A series of 10-fold dilutions in neutralizing broth wasprepared, and plated out in Trypticase Soy Agar. The plates wereincubated at 35° C. for 48 hours, and counted for colony forming units.The results were expressed as:Log reduction=Log initial count of inoculum−Log survivor count on 4hours exposure timeResults

TABLE 1 Log reduction Synthetic example Pseudomonas StaphylococcusEsherichia Fusarium No. aeruginosa aureus coli solani Example 1 6 2.612.25 1.62 0.75 Example 2 7 1.84 5.62 1.84 1.32 Example 3 8 1.72 4.212.01 0.65 Example 4 13 1.51 1.99 2.08 4.49 Example 5 14 2.88 4.18 1.944.49 Comparative 5 0.26 0.14 0.08 0.01 ExampleSurface Modification of Contact Lens with Chitosan DerivativePreparation of Monomer Solution

Trimethylammonium chitosan-g-PEGMA (Synthetic example 6) and PEGDA weredissolved in deionized water (monomer solution). The ratio of monomerwas 2:1 and monomer concentration was 5%. Silicon-containing rigid gaspermeable contact lenses (RGP CLs) were cleaned with deionized water andisopropyl alcohol for 30 minutes in PTFE beaker. And then they weredried under vacuum. The cleaned RGP CLs were treated by argon plasma for60 seconds under a power of 50 W. Argon gas flow rate was 100 sccm. Thenthey were subsequently exposed to the atmosphere for 15 minutes. Theplasma-treated RGP CLs and monomer solution were placed into the glovebox (Argon atmosphere). They were degassed thoroughly. The RGP CLs wereimmersed in a small amount of monomer solution and the top of vial werescrewed tightly. Then they were subjected to UV irradiation (30-35mW/cm²) for 90 minutes. Trimethylammonium chitosan-g-PEGMA coated RGPCLs (Cs-coated lens) were washed thoroughly with deionized water underultrasonication to remove the residual monomer for more than 30 minutes.The results of AFM (Atomic Force Microscope) observation are shown inFIG. 2. FIG. 2 clearly shows that trimethylammonium chitosan-g-PEGMA wascoated on the surface of the RGP CLs.

Antimicrobial Activity

Antimicrobial activity of Cs-coated lens was evaluated in the samemanner as that mentioned above. The results are shown in Table 2.

TABLE 2 Log reduction P. aeruginosa E. coli Cs-coated lens 1.02 2.57Contact Angle

The contact angle was measured by a liquid drop method at roomtemperature by means of contact angle meter Drop Master 500 manufacturedby Kyowa Interface Science Co., Ltd.

-   -   Control (non treated): 93.4°    -   Cs-coated lens: 50.6°

The results reveals that the hydrogel coating of the present inventionexhibits excellent antimicrobial activity and wettability.

What is claimed is:
 1. A quaternized chitosan derivative having thefollowing formula

wherein R′and R″are independently selected from the group consisting ofoptionally substituted C₁-C₁₈alkyl, m is 2 or 3, n is 1 to
 20. 2. Thequaternized chitosan derivative of claim 1, wherein R′has more carbonatoms than R″.
 3. A method of producing the quaternized chitosanderivative of claim 1, comprising a step of reacting a quaternizedchitosan derivative and a halide having a polymerizable organic moiety.4. The method of claim 3, wherein said halide is represented by thefollowing formula:X—CH₂COO(C_(m)H_(2m)O)_(n)COCH═CH₂ wherein X is a halogen atom.